Targeting OXCT1-mediated ketone metabolism reprograms macrophages to promote antitumor immunity via CD8+ T cells in hepatocellular carcinoma.

BACKGROUND & AIMS: The liver is the main organ of ketogenesis, while ketones are mainly metabolized in peripheral tissues via the critical enzyme 3-oxoacid CoA-transferase 1 (OXCT1). We previously found that ketolysis is reactivated in hepatocellular carcinoma (HCC) cells through OXCT1 expression to promote tumor progression; however, whether OXCT1 regulates antitumor immunity remains unclear. METHODS: To investigate the expression pattern of OXCT1 in HCC in vivo, we conducted multiplex immunohistochemistry experiments on human HCC specimens. To explore the role of OXCT1 in mouse HCC tumor-associated macrophages (TAMs), we generated LysMcreOXCT1f/f (OXCT1 conditional knockout in macrophages) mice. RESULTS: Here, we found that inhibiting OXCT1 expression in tumor-associated macrophages reduced CD8+ T-cell exhaustion through the succinate-H3K4me3-Arg1 axis. Initially, we found that OXCT1 was highly expressed in liver macrophages under steady state and that OXCT expression was further increased in TAMs. OXCT1 deficiency in macrophages suppressed tumor growth by reprogramming TAMs toward an antitumor phenotype, reducing CD8+ T-cell exhaustion and increasing CD8+ T-cell cytotoxicity. Mechanistically, high OXCT1 expression induced the accumulation of succinate, a byproduct of ketolysis, in TAMs, which promoted Arg1 transcription by increasing the H3K4me3 level in the Arg1 promoter. In addition, pimozide, an inhibitor of OXCT1, suppressed Arg1 expression as well as TAM polarization toward the protumor phenotype, leading to decreased CD8+ T-cell exhaustion and slower tumor growth. Finally, high expression of OXCT1 in macrophages was positively associated with poor survival in patients with HCC. CONCLUSIONS: In conclusion, our results demonstrate that OXCT1 epigenetically suppresses antitumor immunity, suggesting that suppressing OXCT1 activity in TAMs could be an effective approach for treating liver cancer. IMPACT AND IMPLICATIONS: The intricate metabolism of liver macrophages plays a critical role in shaping hepatocellular carcinoma progression and immune modulation. Targeting macrophage metabolism to counteract immune suppression presents a promising avenue for hepatocellular carcinoma treatment. Herein, we found that the ketogenesis gene OXCT1 was highly expressed in tumor-associated macrophages (TAMs) and promoted tumor growth by reprogramming TAMs toward a protumor phenotype. Pharmacological targeting or genetic downregulation of OXCT1 in TAMs enhances antitumor immunity and slows tumor growth. Our results suggest that suppressing OXCT1 activity in TAMs could be an effective approach for treating liver cancer.

Trace removal of benzene vapour using double-walled metal-dipyrazolate frameworks.

In principle, porous physisorbents are attractive candidates for the removal of volatile organic compounds such as benzene by virtue of their low energy for the capture and release of this pollutant. Unfortunately, many physisorbents exhibit weak sorbate-sorbent interactions, resulting in poor selectivity and low uptake when volatile organic compounds are present at trace concentrations. Herein, we report that a family of double-walled metal-dipyrazolate frameworks, BUT-53 to BUT-58, exhibit benzene uptakes at 298 K of 2.47-3.28 mmol g-1 at <10 Pa. Breakthrough experiments revealed that BUT-55, a supramolecular isomer of the metal-organic framework Co(BDP) (H2BDP = 1,4-di(1H-pyrazol-4-yl)benzene), captures trace levels of benzene, producing an air stream with benzene content below acceptable limits. Furthermore, BUT-55 can be regenerated with mild heating. Insight into the performance of BUT-55 comes from the crystal structure of the benzene-loaded phase (C6H6@BUT-55) and density functional theory calculations, which reveal that C-H···X interactions drive the tight binding of benzene. Our results demonstrate that BUT-55 is a recyclable physisorbent that exhibits high affinity and adsorption capacity towards benzene, making it a candidate for environmental remediation of benzene-contaminated gas mixtures.

Non-Lyn Src Family Kinases Activate SIRPα-SHP-1 to Inhibit PI3K-Akt2 and Dampen Proinflammatory Macrophage Polarization.

Macrophage functional plasticity plays a central role in responding to proinflammatory stimuli. The molecular basis underlying the dynamic phenotypic activation of macrophages, however, remains incompletely understood. In this article, we report that SIRPα is a chief negative regulator of proinflammatory macrophage polarization. In response to TLR agonists, proinflammatory cytokines, or canonical M1 stimulation, Src family kinases (SFK) excluding Lyn phosphorylate SIRPα ITIMs, leading to the preferential recruitment and activation of SHP-1, but not SHP-2. Solely extracellular ligation of SIRPα by CD47 does not greatly induce phosphorylation of SIRPα ITIMs, but it enhances proinflammatory stimuli-induced SIRPα phosphorylation. Examination of downstream signaling elicited by IFN-γ and TLR3/4/9 agonists found that SIRPα-activated SHP-1 moderately represses STAT1, NF-κB, and MAPK signaling but markedly inhibits Akt2, resulting in dampened proinflammatory cytokine production and expression of Ag presentation machinery. Pharmacological inhibition of SHP-1 or deficiency of SIRPα conversely attenuates SIRPα-mediated inhibition and, as such, augments macrophage proinflammatory polarization that in turn exacerbates proinflammation in mouse models of type I diabetes and peritonitis. Our results reveal an SFK-SIRPα-SHP-1 mechanism that fine-tunes macrophage proinflammatory phenotypic activation via inhibition of PI3K-Akt2, which controls the transcription and translation of proinflammatory cytokines, Ag presentation machinery, and other cellular programs.

[Effective substances and mechanism of Yishen Guluo Mixture in treatment of chronic glomerulonephritis based on metabolomics and serum pharmacochemistry].

This study aimed to investigate the effective substances and mechanism of Yishen Guluo Mixture in the treatment of chronic glomerulonephritis(CGN) based on metabolomics and serum pharmacochemistry. The rat model of CGN was induced by cationic bovine serum albumin(C-BSA). After intragastric administration of Yishen Guluo Mixture, the biochemical indexes related to renal function(24-hour urinary protein, serum urea nitrogen, and creatinine) were determined, and the efficacy evaluations such as histopathological observation were carried out. The serum biomarkers of Yishen Guluo Mixture in the treatment of CGN were screened out by ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry(UPLC-Q-TOF-MS) combined with multivariate statistical analysis, and the metabolic pathways were analyzed. According to the mass spectrum ion fragment information and metabolic pathway, the components absorbed into the blood(prototypes and metabolites) from Yishen Guluo Mixture were identified and analyzed by using PeakView 1.2 and MetabolitePilot 2.0.4. By integrating metabolomics and serum pharmacochemistry data, a mathematical model of correlation analysis between serum biomarkers and components absorbed into blood was constructed to screen out the potential effective substances of Yishen Guluo Mixture in the treatment of CGN. Yishen Guluo mixture significantly decreased the levels of 24-hour urinary protein, serum urea nitrogen, and creatinine in rats with CGN, and improved the pathological damage of the kidney tissue. Twenty serum biomarkers of Yishen Guluo Mixture in the treatment of CGN, such as arachidonic acid and lysophosphatidylcholine, were screened out, involving arachidonic acid metabolism, glycerol phosphatide metabolism, and other pathways. Based on the serum pharmacochemistry, 8 prototype components and 20 metabolites in the serum-containing Yishen Guluo Mixture were identified. According to the metabolomics and correlation analysis of serum pharmacochemistry, 12 compounds such as genistein absorbed into the blood from Yishen Guluo Mixture were selected as the potential effective substances for the treatment of CGN. Based on metabolomics and serum pharmacochemistry, the effective substances and mechanism of Yishen Guluo Mixture in the treatment of CGN are analyzed and explained in this study, which provides a new idea for the development of innovative traditional Chinese medicine for the treatment of CGN.

Inflammation Unrestrained by SIRPα Induces Secondary Hemophagocytic Lymphohistiocytosis Independent of IFN-γ.

A hallmark of secondary hemophagocytic lymphohistiocytosis (sHLH), a severe form of cytokine storm syndrome, is the emergence of overactivated macrophages that engulf healthy host blood cells (i.e., hemophagocytosis) and contribute to the dysregulated inflammation-driven pathology. In this study, we show that depleting SIRPα (SIRPα-/-) in mice during TLR9-driven inflammation exacerbates and accelerates the onset of fulminant sHLH, in which systemic hemophagocytosis, hypercytokinemia, consumptive cytopenias, hyperferritinemia, and other hemophagocytic lymphohistiocytosis hallmarks were apparent. In contrast, mice expressing SIRPα, including those deficient of the SIRPα ligand CD47 (CD47-/-), do not phenocopy SIRPα deficiency and fail to fully develop sHLH, albeit TLR9-inflamed wild-type and CD47-/- mice exhibited hemophagocytosis, anemia, and splenomegaly. Although IFN-γ is largely considered a driver of hemophagocytic lymphohistiocytosis pathology, IFN-γ neutralization did not preclude the precipitation of sHLH in TLR9-inflamed SIRPα-/- mice, whereas macrophage depletion attenuated sHLH in SIRPα-/- mice. Mechanistic studies confirmed that SIRPα not only restrains macrophages from acquiring a hemophagocytic phenotype but also tempers their proinflammatory cytokine and ferritin secretion by negatively regulating Erk1/2 and p38 activation downstream of TLR9 signaling. In addition to TLR9 agonists, TLR2, TLR3, or TLR4 agonists, as well as TNF-α, IL-6, or IL-17A, but not IFN-γ, similarly induced sHLH in SIRPα-/- mice but not SIRPα+ mice. Collectively, our study suggests that SIRPα plays a previously unappreciated role in sHLH/cytokine storm syndrome pathogenesis by preventing macrophages from becoming both hemophagocytic and hyperactivated under proinflammation.

A novel highly specific colorimetric fluorescent probe for the detection of nitrite in aqueous solution.

Developing an effective method for the detection of nitrite (NO2 - ) ions in the natural environment especially in environmental waters and soils is very necessary, since they will cause serious damage to human health once excess NO2 - ions enters the human body. Therefore, a new colorimetric fluorescent probe NB-NO2 - for determining NO2 - ions was designed, which possesses good water-solubility and satisfactory selectivity over other common ions for NO2 - ions. The addition of NO2 - ions changed the color of solution from blue to colorless seen by the naked-eye. Furthermore, through test and calculation, the detection limit of the probe NB-NO2 - is 129 nM. Based on the earlier excellent characteristics, the probe NB-NO2 - was successfully used for monitoring NO2 - ions in environmental waters and soils.

Novel Minimally-Invasive Triple Pelvic Osteotomy: JiShuiTan Minimally-Invasive Approach.

PURPOSE: Triple pelvic osteotomy (TPO) is often performed to improve femoral head coverage, correct deformity, and stabilize the hip joint in a variety of pediatric orthopaedic conditions. After the TPO was first reported, many modifications were developed to simplify or improve the procedure, however, because of the specific anatomy with several critical nerves and vessels passing through the approaches, extensive exposure and prolonged intraoperative fluoroscopy are often required for TPO. This report introduces a novel, minimally-invasive surgical approach that minimizes the time of intraoperative fluoroscopy and size of the surgical incision, and reviews our experience. METHODS: A total of 48 hips in 43 patients with a mean age of 8.3±1.7 years (range: 6.0 to 12.2 y) were included in this study. Of these, 21 patients (22 hips) had Legg-Calvé-Perthes disease (LCPD) and 22 patients (26 hips) had developmental dysplasia of the hip (DDH). The TPOs were performed using the novel, minimally-invasive TPO approach, with a mean postoperative follow-up of 38 months (range: 24 to 54 mo) in the DDH group and 44 months (range: 23 to 58 mo) in the LCPD group. The acetabular index (AI), femoral head migration rate (MP), center edge angle (CEA) and the Severin and Stulberg classification systems were used to evaluate the preoperative and postoperative results. SPSS software was used to analyze the data. RESULTS: The AI decreased from 33.8 degrees±9.2 to 2.9 degrees±10.1, the lateral CEA increased from -10.8 degrees±23.8 to 34.1 degrees±9.9, and the femoral head MP decreased from 64.0%±19.8% to 1.1%±2.6% in the DDH group at last follow-up, indicating significant improvement. The AI decreased from 20.8 degrees±4.7 to -1.3 degrees±7.3, the lateral CEA increased from 6.8 degrees±11.5 to 42.3 degrees±6.4, and the femoral head MP decreased from 42.2%±13.0% to 1.3%±3.3% in the LCPD group at last follow-up, also indicating significant improvement. CONCLUSION: This approach can simplify the TPO, making the complex operation safer, more effective, and capable of achieving satisfactory correction.

Risk Factors for Avascular Necrosis After Closed Reduction for Developmental Dysplasia of the Hip.

BACKGROUND: Avascular necrosis (AVN) is a major complication after closed reduction for developmental dysplasia of the hip. The factors that predispose to AVN remain controversial. The purpose of this study was to analyze the risk factors, especially patient factors, such as age at reduction, grade of dislocation, and ossific nucleus development, related to AVN. MATERIALS AND METHODS: We retrospectively reviewed children with dysplasia of the hip treated by closed reduction between 1997 and 2006. AVN was evaluated using Salter criteria and Kalamchi and MacEwen classification. Related factors were analyzed. RESULTS: One hundred and eight children (140 hips) with an average age of 16.6 months at closed reduction (range: 6-24 mo) were included in the study. For an average duration of 10.1 years (range 7-16 y) of follow-up, 44 hips (31.4%) developed AVN. Grade II or higher AVN occurred in 14 hips (10%). The incidence of AVN increased with the grade of dislocation ( P =0.022) and underdevelopment of the ossific nucleus ( P <0.001). Underdevelopment of the ossific nucleus was also found to be positively correlated with the dislocation grade ( P =0.047). The age at the time of reduction, sex, and side were not significant factors. Children who underwent secondary operation were all older than 1 year at reduction. CONCLUSIONS: High-grade dislocation correlates with the underdevelopment of the ossific nucleus. Patients with these 2 characteristics are predisposed to AVN. As underdevelopment of the ossific nucleus occurred regardless of age, it is not advisable to delay reduction because it does not alter the AVN rate, and instead, it increases the secondary operation rate. LEVEL OF EVIDENCE: Level IV case series.

[Prediction of material basis and mechanism of Curcumae Rhizoma in treatment of coronary heart disease].

Coronary heart disease(CHD) is a common cardiovascular disease in clinical practice. Curcumae Rhizoma(CR), an important herbal medicine for breaking blood stasis and resolving mass, is often used for the treatment of CHD caused by blood stasis syndrome. However, the anti-CHD components, targets, and mechanism are still unclear. Therefore, in this study, the chemical components of CR were separated and identified by ultra high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS). Based on the identified components, network pharmacology analysis, including target prediction and functional enrichment, was applied to screen out the main active components against CHD, and the potential mechanism was discussed. Finally, molecular docking was performed to verify the binding between the active components and the targets. The results showed that among the 52 chemical components identified in CR, 28 were related to CHD, involving 75 core targets. The core components included(4S)-4-hydroxy-gweicurculactone, curcumadione, and curcumenone, and the core targets included phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha(PIK3 CA), mitogen-activated protein kinase 1(MAPK1), and mitogen-activated protein kinase 3(MAPK3). In summary, through the active components, such as(4S)-4-hydroxy-gweicurculactone, curcumadione, and curcumenone, CR regulates the nerve repair, vasoconstriction, lipid metabolism, and inflammatory response, thereby exerts therapeutic effect on CHD.

[Effective substance and mechanism of Ziziphi Spinosae Semen extract in treatment of insomnia based on serum metabolomics and network pharmacology].

This study aims to study the effective substance and mechanism of Ziziphi Spinosae Semen extract in the treatment of insomnia based on serum metabolomics and network pharmacology. The rat insomnia model induced by p-chlorophenylalanine(PCPA) was established. After oral administration of Ziziphi Spinosae Semen extract, the general morphological observation, pentobarbital sodium-induced sleep test, and histopathological evaluation were carried out. The potential biomarkers of the extract in the treatment of insomnia were screened by ultra-high performance liquid chromatography-mass spectrometry(UHPLC-MS) combined with multivariate analysis, and the related metabolic pathways were further analyzed. The "component-target-pathway" network was constructed by ultra-high performance liquid chromatography coupled with quadrupole-Exactive mass spectrometry(UHPLC-Q-Exactive-MS/MS) combined with network pharmacology to explore the effective substances and mechanism of Ziziphi Spinosae Semen in the treatment of insomnia. The results of pentobarbital sodium-induced sleep test and histopathological evaluation(hematoxylin and eosin staining) showed that Ziziphi Spinosae Semen extract had good theraputic effect on insomnia. A total of 21 endogenous biomarkers of Ziziphi Spinosae Semen extract in the treatment of insomnia were screened out by serum metabolomics, and the metabolic pathways of phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, and nicotinate and nicotinamide metabolism were obtained. A total of 34 chemical constituents were identified by UHPLC-Q-Exactive-MS/MS, including 24 flavonoids, 2 triterpenoid saponins, 4 alkaloids, 2 triterpenoid acids, and 2 fatty acids. The network pharmacological analysis showed that Ziziphi Spinosae Semen mainly acted on target proteins such as dopamine D2 receptor(DRD2), 5-hydroxytryptamine receptor 1 A(HTR1 A), and alpha-2 A adrenergic receptor(ADRA2 A) in the treatment of insomnia. It was closely related to neuroactive ligand-receptor interaction, serotonergic synapse, and calcium signaling pathway. Magnoflorine, N-nornuciferine, caaverine, oleic acid, palmitic acid, coclaurine, betulinic acid, and ceanothic acid in Ziziphi Spinosae Semen may be potential effective compounds in the treatment of insomnia. This study revealed that Ziziphi Spinosae Semen extract treated insomnia through multiple metabolic pathways and the overall correction of metabolic disorder profile in a multi-component, multi-target, and multi-channel manner. Briefly, this study lays a foundation for further research on the mechanism of Ziziphi Spinosae Semen in treating insomnia and provides support for the development of innovative Chinese drugs for the treatment of insomnia.

Trans-olecranon fracture-dislocation of the elbow in children.

OBJECTIVES: Trans-olecranon fracture-dislocations are rare in children. To our knowledge, only 12 cases have been described in children till now and the treatment strategy for this injury in children remains unclear. To provide a clear clinical description and more accurate treatment options, we retrospectively reviewed cases with this kind of injury in our institution. METHODS: From 2002 to 2019, eleven cases diagnosed with trans-olecranon fracture-dislocation of the elbow were identified, and their medical charts and radiographs were obtained. All patients underwent open reduction and internal fixation through a posterior approach. At the most recent follow-up visit, all patients were evaluated clinically using the Mayo Elbow Performance Score (MEPS). RESULTS: The mean follow-up was 22 months (range, 6-42 months). All injuries were unilateral, and there were nine males and two females. The mean age at injury was nine years (range, 4-13 years), and the mean time from injury to surgery was 16.6 days (range, 2-60 days). According to Tiemdjo classification, there was one case with type I injury, one case with type II, six cases with type III, and three cases with type IV. According to the MEPS criteria, the outcomes were excellent in five cases, good in two cases, fair in one case, and poor in three cases. Four patients were delayed cases, who underwent surgery two weeks after injury. The average operation time was significantly longer in four children sustaining delayed surgery (140 ± 43 min, vs. 50 ± 12 min, p < 0.001). CONCLUSION: To our knowledge, this is the largest sample size reported to date. We recommend open reduction and internal fixation, using either plates or tension-band techniques, depending on the injury pattern. In addition, we emphasize that early operation could achieve good clinical outcomes.

Tumor conditions induce bone marrow expansion of granulocytic, but not monocytic, immunosuppressive leukocytes with increased CXCR2 expression in mice.

Myeloid-derived suppressor cells (MDSCs) promote tumor growth through, in part, inhibiting T-cell immunity. However, mechanisms underlying MDSC expansion and guidance of MDSCs toward the tumor microenvironment remain unclear. Employing Percoll density gradients, we separate bone marrow (BM) leukocytes from tumor-bearing mice into four density-increasing bands with myeloid leukocytes enriched in bands III and IV. Band III comprises monocytes and low-density granulocytes, both confirmed to be M-MDSCs and G-MDSCs, respectively, by displaying potent inhibition of T-cell proliferation. However, monocytes act as M-MDSCs not only under tumor conditions but also the healthy condition. In contrast, band IV contains non-inhibitory, mature granulocytes. Only band III G-MDSCs display significant expansion in mice bearing B16 melanoma, Lewis lung carcinoma, or MC38 colon carcinoma. The expanded G-MDSCs also show increased CXCR2 expression, which guides egress out of BM, and produce arginase-1 and ROS upon encountering antigen-activated T cells. Adoptive transfer assays demonstrate that both G-MDSCs and mature granulocytes infiltrate tumors, but only the former displays sustention and accumulation. Intratumoral administrations of granulocytes further demonstrate that G-MDSCs promote tumor growth, whereas mature granulocytes exert minimal effects, or execute powerful anti-tumor effects providing the presence of PMN activation mechanisms in the tumor microenvironment.

Arginase-1 is neither constitutively expressed in nor required for myeloid-derived suppressor cell-mediated inhibition of T-cell proliferation.

Although previous reports suggest that tumor-induced myeloid-derived suppressor cells (MDSC) inhibit T cells by L-arginine depletion through arginase-1 activity, we herein show that arginase-1 is neither inherently expressed in MDSC nor required for MDSC-mediated inhibition. Employing Percoll density gradients, large expansions of MDSC in the bone marrow of tumor-bearing mice were isolated and demonstrated potent inhibition in T-cell proliferation activated by TCR-ligation, Concanavalin A, PMA plus ionomycin, or IL-2. Despite demonstrating characteristic immunosuppressive capacity, these MDSC exhibit no arginase-1 expression and/or exert their inhibitory effects independent of arginase-1 activity. However, arginase-1 expression in MDSC can be induced by exposure to TCR-activated T cells or their culture medium, but not T cells activated by other means or growing tumor cells. Further investigation reveals multiple cytokines secreted by TCR-activated T cells as orchestrating two signaling-relay axes, IL-6-to-IL-4 and GM-CSF/IL-4-to-IL-10, leading to arginase-1 expression in MDSC. Specifically, IL-6 signaling increases IL-4R, enabling IL-4 to induce arginase-1 expression; similarly, GM-CSF in concert with IL-4 induces IL-10R, allowing IL-10-mediated induction. Surprisingly, our study indicates that induction of arginase-1 expression is not conducive to the critical MDSC-mediated inhibition toward T cells, which is rather dependent on direct cell contacts undiminished by PD-L1 blockade or SIRPα deficiency.

Immune checkpoint molecules. Possible future therapeutic implications in autoimmune diseases.

During host immune response, an initial and sufficient activation is required to avoid infection and cancer, yet an excessive activation bears the risk of autoimmune reactivity and disease development. This fastidious balance of the immune system is regulated by co-stimulatory and co-inhibitory molecules, also known as immune checkpoints. Both excessive co-stimulation and insufficient co-inhibition can induce the activation and proliferation of autoreactive cells that may lead to the development of autoimmune diseases. During the last decade, a growing number of new immune checkpoint receptors and ligands have been discovered, providing an attractive approach to investigate their implication in the pathogenesis of autoimmune diseases and their potential role as targets for effective therapeutic interventions. In this review, we focus on the roles and underlying mechanisms of co-stimulatory and co-inhibitory receptors and other molecules that function as immune checkpoints in autoimmune diseases such as systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, Sjögren's syndrome, type I diabetes and inflammatory bowel disease. We also summarize previous and current clinical trials targeting these checkpoint pathways in autoimmune diseases and discuss further therapeutic implications and possible risks and challenges.

Cd47-Sirpα interaction and IL-10 constrain inflammation-induced macrophage phagocytosis of healthy self-cells.

Rapid clearance of adoptively transferred Cd47-null (Cd47(-/-)) cells in congeneic WT mice suggests a critical self-recognition mechanism, in which CD47 is the ubiquitous marker of self, and its interaction with macrophage signal regulatory protein α (SIRPα) triggers inhibitory signaling through SIRPα cytoplasmic immunoreceptor tyrosine-based inhibition motifs and tyrosine phosphatase SHP-1/2. However, instead of displaying self-destruction phenotypes, Cd47(-/-) mice manifest no, or only mild, macrophage phagocytosis toward self-cells except under the nonobese diabetic background. Studying our recently established Sirpα-KO (Sirpα(-/-)) mice, as well as Cd47(-/-) mice, we reveal additional activation and inhibitory mechanisms besides the CD47-SIRPα axis dominantly controlling macrophage behavior. Sirpα(-/-) mice and Cd47(-/-) mice, although being normally healthy, develop severe anemia and splenomegaly under chronic colitis, peritonitis, cytokine treatments, and CFA-/LPS-induced inflammation, owing to splenic macrophages phagocytizing self-red blood cells. Ex vivo phagocytosis assays confirmed general inactivity of macrophages from Sirpα(-/-) or Cd47(-/-) mice toward healthy self-cells, whereas they aggressively attack toward bacteria, zymosan, apoptotic, and immune complex-bound cells; however, treating these macrophages with IL-17, LPS, IL-6, IL-1ß, and TNFα, but not IFNγ, dramatically initiates potent phagocytosis toward self-cells, for which only the Cd47-Sirpα interaction restrains. Even for macrophages from WT mice, phagocytosis toward Cd47(-/-) cells does not occur without phagocytic activation. Mechanistic studies suggest a PKC-Syk-mediated signaling pathway, to which IL-10 conversely inhibits, is required for activating macrophage self-targeting, followed by phagocytosis independent of calreticulin Moreover, we identified spleen red pulp to be one specific tissue that provides stimuli constantly activating macrophage phagocytosis albeit lacking in Cd47(-/-) or Sirpα(-/-) mice.

Broad Infiltration of Macrophages Leads to a Proinflammatory State in Streptozotocin-Induced Hyperglycemic Mice.

Chronic diseases are often associated with altered inflammatory response, leading to increased host vulnerability to new inflammatory challenges. Employing streptozotocin (STZ)-induced diabetes as a model, we further investigate mechanisms leading to enhanced neutrophil (polymorphonuclear leukocytes [PMN]) responses under hyperglycemia and compare them with those under chronic colitis. We show that, different from colitis under which the PMN response is significantly potentiated, the existence of a proinflammatory state associated with broad increases in macrophages in various organs plays a dominant role in promoting the PMN inflammatory response in diabetic mice. Studies of PMN infiltration during zymosan-induced peritonitis reveal that hyperglycemia enhances PMN recruitment not through inducing a high level of IL-17, which is the case in colitis, but through increasing F4/80+ macrophages in the peritoneal cavity, resulting in elevations of IL-6, IL-1ß, TNF-α, and CXCL1 production. Insulin reversal of hyperglycemia, but not the neutralization of IL-17, reduces peritoneal macrophage numbers and ameliorates PMN infiltration during peritonitis. Significantly increased macrophages are also observed in the liver, kidneys, and intestines under hyperglycemia, and they are attributable to exacerbated nephropathy and colitis when inflammatory conditions are induced by doxorubicin and dextran sulfate sodium, respectively. Furthermore, analyses of monocyte production and macrophage proliferation in tissues suggest that significant monocytosis of inflammatory F4/80+Gr-1+ monocytes from the spleen and macrophage proliferation in situ synergistically contribute to the increased macrophage population under hyperglycemia. In conclusion, our results demonstrate that STZ-induced hyperglycemic mice develop a systemic proinflammatory state mediated by broad infiltration of macrophages.

Secreted monocytic miR-150 enhances targeted endothelial cell migration.

MicroRNAs (miRNAs) are a class of noncoding RNAs that regulate target gene expression at the posttranscriptional level. Here, we report that secreted miRNAs can serve as signaling molecules mediating intercellular communication. In human blood cells and cultured THP-1 cells, miR-150 was selectively packaged into microvesicles (MVs) and actively secreted. THP-1-derived MVs can enter and deliver miR-150 into human HMEC-1 cells, and elevated exogenous miR-150 effectively reduced c-Myb expression and enhanced cell migration in HMEC-1 cells. In vivo studies confirmed that intravenous injection of THP-1 MVs significantly increased the level of miR-150 in mouse blood vessels. MVs isolated from the plasma of patients with atherosclerosis contained higher levels of miR-150, and they more effectively promoted HMEC-1 cell migration than MVs from healthy donors. These results demonstrate that cells can secrete miRNAs and deliver them into recipient cells where the exogenous miRNAs can regulate target gene expression and recipient cell function.

CD47 Plays a Role as a Negative Regulator in Inducing Protective Immune Responses to Vaccination against Influenza Virus.

UNLABELLED: An integrin-associated protein CD47, which is a ligand for the inhibitory receptor signal regulatory protein α, is expressed on B and T cells, as well as on most innate immune cells. However, the roles of CD47 in the immune responses to viral infection or vaccination remain unknown. We investigated the role of CD47 in inducing humoral immune responses after intranasal infection with virus or immunization with influenza virus-like particles (VLPs). Virus infection or vaccination with VLPs containing hemagglutinin from A/PR8/34 influenza virus induced higher levels of antigen-specific IgG2c isotype dominant antibodies in CD47-deficient (CD47KO) mice than in wild-type (WT) mice. CD47KO mice with vaccination showed greater protective efficacy against lethal challenge, as evidenced by no loss in body weight and reduced lung viral titers compared to WT mice. In addition, inflammatory responses which include cytokine production, leukocyte infiltrates, and gamma interferon-producing CD4(+) T cells, as well as an anti-inflammatory cytokine (interleukin-10), were reduced in the lungs of vaccinated CD47KO mice after challenge with influenza virus. Analysis of lymphocytes indicated that GL7(+) germinal center B cells were induced at higher levels in the draining lymph nodes of CD47KO mice compared to those in WT mice. Notably, CD47KO mice exhibited significant increases in the numbers of antigen-specific memory B cells in spleens and plasma cells in bone marrow despite their lower levels of background IgG antibodies. These results suggest that CD47 plays a role as a negative regulator in inducing protective immune responses to influenza vaccination. IMPORTANCE: Molecular mechanisms that control B cell activation to produce protective antibodies upon viral vaccination remain poorly understood. The CD47 molecule is known to be a ligand for the inhibitory receptor signal regulatory protein α and expressed on the surfaces of most immune cell types. CD47 was previously demonstrated to play an important role in modulating the migration of monocytes, neutrophils, polymorphonuclear neutrophils, and dendritic cells into the inflamed tissues. The results of this study demonstrate new roles of CD47 in negatively regulating the induction of protective IgG antibodies, germinal center B cells, and plasma cells secreting antigen-specific antibodies, as well as macrophages, upon influenza vaccination and challenge. As a consequence, vaccinated CD47-deficient mice demonstrated better control of influenza viral infection and enhanced protection. This study provides insights into understanding the regulatory functions of CD47 in inducing adaptive immunity to vaccination.

Loss of Cell Surface CD47 Clustering Formation and Binding Avidity to SIRPα Facilitate Apoptotic Cell Clearance by Macrophages.

CD47, a self recognition marker expressed on tissue cells, interacts with immunoreceptor SIRPα expressed on the surface of macrophages to initiate inhibitory signaling that prevents macrophage phagocytosis of healthy host cells. Previous studies suggested that cells may lose surface CD47 during aging or apoptosis to enable phagocytic clearance. In the current study, we demonstrate that the level of cell surface CD47 is not decreased, but the distribution pattern of CD47 is altered, during apoptosis. On nonapoptotic cells, CD47 molecules are clustered in lipid rafts forming punctates on the surface, whereas on apoptotic cells, CD47 molecules are diffused on the cell surface following the disassembly of lipid rafts. We show that clustering of CD47 in lipid rafts provides a high binding avidity for cell surface CD47 to ligate macrophage SIRPα, which also presents as clusters, and elicits SIRPα-mediated inhibitory signaling that prevents phagocytosis. In contrast, dispersed CD47 on the apoptotic cell surface is associated with a significant reduction in the binding avidity to SIRPα and a failure to trigger SIRPα signal transduction. Disruption of plasma membrane lipid rafts with methyl-ß-cyclodextrin diffuses CD47 clusters, leading to a decrease in the cell binding avidity to SIRPα and a concomitant increase in cells being engulfed by macrophages. Taken together, our study reveals that CD47 normally is clustered in lipid rafts on nonapoptotic cells but is diffused in the plasma membrane when apoptosis occurs; this transformation of CD47 greatly reduces the strength of CD47-SIRPα engagement, resulting in the phagocytosis of apoptotic cells.

CD47 deficiency ameliorates autoimmune nephritis in Fas(lpr) mice by suppressing IgG autoantibody production.

CD47, a self-recognition marker, plays an important role in both innate and adaptive immune responses. To explore the potential role of CD47 in activation of autoreactive T and B cells and the production of autoantibodies in autoimmune disease, especially systemic lupus erythematosus (SLE), we have generated CD47 knockout Fas(lpr) (CD47(-/-) -Fas(lpr) ) mice and examined histopathological changes in the kidneys, cumulative survival rates, proteinuria, extent of splenomegaly and autoantibodies, serum chemistry and immunological parameters. In comparison with Fas(lpr) mice, CD47(-/-) -Fas(lpr) mice exhibit a prolonged lifespan and delayed autoimmune nephritis, including glomerular cell proliferation, basement membrane thickening, acute tubular atrophy and vacuolization. CD47(-/-) -Fas(lpr) mice have lower levels of proteinuria, associated with reduced deposition of complement C3 and C1q, and IgG but not IgM in the glomeruli, compared to age-matched Fas(lpr) mice. Serum levels of antinuclear antibodies and anti-double-stranded DNA antibodies are significantly lower in CD47(-/-) -Fas(lpr) than in Fas(lpr) mice. CD47(-/-) -Fas(lpr) mice also display less pronounced splenomegaly than Fas(lpr) mice. The mechanistic studies further suggest that CD47 deficiency impairs the antigenic challenge-induced production of IgG but not IgM, and that this effect is associated with reduction of T follicular cells and impairment of germinal centre development in lymphoid tissues. In conclusion, our results demonstrate that CD47 deficiency ameliorates lupus nephritis in Fas(lpr) mice via suppression of IgG autoantibody production.